Magnetic molecularly imprinted polymers for extraction of S-phenylmercapturic acid from urine samples followed by high-performance liquid chromatography.

In this study, magnetic molecularly imprinted polymers (MMIPs) were prepared and used as sorbents for extraction of S-phenylmercapturic acid (S-PMA) from urine samples, followed by high-performance liquid chromatography ultraviolet-visible (HPLC-UV/Vis) analysis. The MMIPs were synthesized by the copolymerization reaction of (phenylthio) acetic acid (template molecule), methacrylic acid (functional monomers) and ethylene glycol dimethacrylate (cross-linkers). The morphology, structure property and surface groups of the prepared MMIPs were characterized by scan electron microscopy, transmission electron microscopy, infrared spectroscopy, X-ray diffraction pattern, thermogravimetric analyses, Brunauer-Emmett-Teller and vibrating sample magnetometer. The selectivity of the MMIPs was investigated in the presence of interferents. Various parameters affecting the S-PMA extraction efficiency were investigated, including MMIPs amount, pH, sample volume, desorption solvent, as well as extraction and desorption time. The obtained optimal parameters were as follows: MMIPs amount (20 mg), pH (3.0), sample volume (5 mL), desorption solvent (methanol/acetic acid [9/1, v/v]), extraction time (30 minutes) and desorption time (2 minutes). The method was validated according to the Food and Drug Administration Guidance for Industry on Bioanalytical Method Validation. The calibration curve for the analyte was linear in the concentration range of 0.030-1.0 mg/L (r = 0.9995). The LOD and LOQ of the method were 0.0080 and 0.0267 mg/L, respectively. The enrichment factor of the MMIPs was 5. The relative standard deviations of intra- and inter-day tests were in the range of 3.8-5.1% and 3.9-6.3%, respectively. The recoveries at three different concentrations of 0.10, 0.50 and 0.80 mg/L ranged between 95.2% and 98.6%. In addition, the MMIPs could be reused for at least eight times. The proposed method was successfully applied to the determination of S-PMA in urine samples. In addition, this developed method could be used as a tool in the early screening and clinical diagnosis of benzene intoxication.

Activated carbon/diatomite-based magnetic nanocomposites for magnetic solid-phase extraction of S-phenylmercapturic acid from human urine.

In this study, activated carbon/diatomite-based magnetic nancomposites (denoted as AC/DBMNs) were synthesized and applied as an adsorbent for magnetic solid-phase extraction of S-phenylmercapturic acid (S-PMA) from human urine prior to high-performance liquid chromatography. The surface morphologies and structures of AC/DBMNs were characterized by Fourier transform infrared spectroscopy, transmission and scanning electron microscopy, X-ray diffraction, Brunauer-Emmett-Teller surface area, vibrating sample magnetometer and ζ-potential measurements. The experimental parameters including sample volume, sample pH, adsorbent amount, extraction time, elution solvent and desorption time were investigated in detail. Under the optimum conditions, the method exhibited good linearity (r > 0.9993) within the concentration ranges of 0.03-1.0 mg/L. Moreover, the limits of detection and quantification were 0.01 and 0.03 mg/L, respectively. The enrichment factor was 5, and good recoveries (88.9-97.3%) with relative standard deviations in the range of 5.6-6.8% (n = 6) for inter-day and 6.3-8.1% (n = 6) for intra-day were achieved. The developed method was successfully applied to the analysis of S-PMA in urine samples. In addition, this accurate and sensitive method has great potential to be applied in the early screening and clinical diagnosis of the workers exposed to benzene.

Lactacystin: first-in-class proteasome inhibitor still excelling and an exemplar for future antibiotic research.

Lactacystin exemplifies the role that serendipity plays in drug discovery and why "finding things without actually looking for them" retains such a pivotal role in the search for the useful properties of chemicals. The first proteasome inhibitor discovered, lactacystin stimulated new possibilities in cancer control. New and innovative uses are regularly being found for lactacystin, including as a model to study dementia, while new formulations and delivery systems may facilitate its use clinically as an anticancer agent. All this provides yet more evidence that we need a comprehensive, collaborative and coordinated programme to fully investigate all new and existing chemical compounds, especially those of microbial origin. We need to do so in order to avoid failing to detect and successfully exploit unsought yet potentially life-saving or extremely advantageous properties of microbial metabolites.

A Novel Natural Product-Derived Compound, Vestaine A1, Exerts both Pro-Angiogenic and Anti-Permeability Activity via a Different Pathway from VEGF.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is a key molecule in the regulation of both angiogenesis and vascular permeability. However, it is known that overproduction of VEGF induces abnormal blood vessel formation and these vessels cause several disease pathologies, such as diabetic retinopathy. The purpose of this study was to find novel vasoactive compounds which have different properties from VEGF. METHODS/RESULTS: We screened a natural product library using a co-culture angiogenic assay of endothelial cells and fibroblasts. By focusing on morphological changes of endothelial cells, we isolated the novel compounds vestaine A1 and vestaine B1 from the cultured broth of an actinomycete strain, Streptomyces sp. SANK 63697. Vestaine A1 enhanced tube formation of endothelial cells in Matrigel and suppressed cell death induced by serum deprivation. Vestaine A1 activated both MEK1/2 and PI-3 kinase pathways independently of the VEGF pathway in a dose- and time-dependent fashion. Finally, vestaine A1 potently suppressed VEGF-induced vascular permeability both in vitro and in vivo. CONCLUSION: Vestaine A1 has the potential to exhibit both pro-angiogenic and anti-permeability properties, and would therefore be useful for therapeutic treatment for abnormal vascular permeability-related diseases.

Recovery by N-acetylcysteine from subchronic exposure to Imidacloprid-induced hypothalamic-pituitary-adrenal (HPA) axis tissues injury in male rats.

Imidacloprid is the most important example of the neonicotinoid insecticides known to target the nicotinic acetylcholine receptor in insects, and potentially in mammals. N-Acetyl-l-cysteine (NAC) has been shown to possess curative effects in experimental and clinical investigations. The present study was designed to evaluate the recovery effect of NAC against Imidacloprid-induced oxidative stress and cholinergic transmission alteration in hypothalamic-pituitary-adrenal (HPA) axis of male rats following subchronic exposure. About 40 mg/kg of Imidacloprid was administered daily by intragastric intubation and 28 days later, the rats were sacrificed and HPA axis tissues were removed for different analyses. Imidacloprid increased adrenal relative weight and cholesterol level indicating an adaptive stage of the general alarm reaction to stress. Moreover, Imidacloprid caused a significant increase in malondialdehyde level, the antioxidants catalase, superoxide dismutase and glutathione-S-transferase showed various alterations following administration and significant depleted thiols content was only recorded in hypothalamic tissue. Furthermore, the hypothalamic and pituitary acetylcholinesterase activity and calcium level were significantly increased highlighting the alteration of cholinergic activity. The present findings revealed that HPA axis is a sensitive target to Imidacloprid (IMI). Interestingly, the use of NAC for only 7 days post-exposure to IMI showed a partial therapeutic effect against Imidacloprid toxicity.

A new BODIPY-based long-wavelength fluorescent probe for chromatographic analysis of low-molecular-weight thiols.

A new long-wavelength fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene (DMDSPAB-I), was designed and synthesized for thiol labeling in high-performance liquid chromatography (HPLC). The excitation and emission wavelengths of DMDSPAB-I are 620 and 630 nm, respectively, with a high fluorescence quantum yield of 0.557, which is advantageous in preventing interference of intrinsic fluorescence from complex biological matrices and enabling high sensitivity HPLC. Based on DMDSPAB-I, a reversed-phase HPLC method was developed for measuring low-molecular-weight thiols including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. After the specific reaction of DMDSPAB-I with thiols, baseline separation of all six stable derivatives was achieved through isocratic elution on a C18 column within 25 min, with the limits of detection (signal-to-noise ratio = 3) from 0.24 nmol L(-1) for glutathione to 0.72 nmol L(-1) for penicillamine. The proposed method was validated in part by measuring thiols in blood samples from mice, with recoveries of 95.3-104.3%.

Farnesol in combination with N-acetylcysteine against Staphylococcus epidermidis planktonic and biofilm cells

Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections, in which biofilm formation is considered to be the main virulence mechanism. In biofilm environment, microbes exhibit enhanced resistance to antimicrobial agents. This fact boosted the search of possible alternatives to antibiotics. Farnesol and N-acetylcysteine (NAC) are non-antibiotic drugs that have demonstrated antibacterial properties. In this study, the effect of farnesol and NAC isolated or in combination (farnesol+NAC) was evaluated. NAC at 10 × MIC caused a total cell death in planktonic cells. On the other hand, S. epidermidis biofilms exhibited 4 log reduction in viable cell number after a 24h treatment with NAC at the former concentration. Our results demonstrated that there was a higher CFU log reduction of S. epidermidis planktonic cells when farnesol was combined with NAC at 1 × MIC relatively to each agent alone. However, these results were not relevant because NAC alone at 10 × MIC was always the condition which gave the best results, having a very high killing effect on planktonic cells and a significant bactericidal effect on biofilm cells. This study demonstrated that no synergy was observed between farnesol and NAC. However, the pronounced antibacterial effect of NAC against S. epidermidis, on both lifestyles, indicates the use of NAC as a potential therapeutic agent in alternative to antibiotics.

Cyslabdan, a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus, produced by Streptomyces sp. K04-0144. I. Taxonomy, fermentation, isolation and structural elucidation.

Cyslabdan, a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus, was isolated from the culture broth of Streptomyces sp. K04-0144 by Diaion HP-20 and ODS column chromatographies and preparative HPLC. The structure of cyslabdan was elucidated by spectroscopic analyses including NMR. The compound has a labdane-type diterpene skeleton connecting with an N-acetylcysteine via thioether linkage.

Cinnabaramides A-G: analogues of lactacystin and salinosporamide from a terrestrial streptomycete.

The cinnabaramides A-G (1-7) were isolated from a terrestrial strain of Streptomyces as potent and selective inhibitors of the human 20S proteasome. Their chemical and biological properties resemble those of salinosporamide A, a recently identified lead compound from an obligate marine actinomycete, which is currently under development as an anticancer agent. Cinnabaramides F and G (6, 7) combine essential structural features of salinosporamide A and lactacystin and show about equal potency in vitro, with IC50 values in the 1 nM range. The properties and phylogenetic position of the producer organism, the production and isolation of compounds 1-7, their structure elucidation by MS and NMR, and their biological activities are reported. Additionally, an X-ray crystal structure was obtained from cinnabaramide A (1).

Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection.

N-acetyl-l-cysteine (NAC) is a well-known antioxidant that is capable of facilitating glutathione (GSH) biosynthesis and replenishing intracellular GSH under oxidatively challenging circumstances. N-acetyl-cysteine-amide (NACA), the amide form of NAC, is a newly designed and synthesized thiol-containing compound which is believed to be more lipophilic and permeable through cell membranes than NAC. The metabolic and antioxidant effects of these compounds in vitro and in vivo are under investigation. However, an analytical method that can separate and quantify both compounds simultaneously is not yet available, to the best of our knowledge. Because of their structural similarities, the two compounds are difficult to separate using earlier HPLC methods which were designed for NAC quantification. Therefore, the goal of this work was to develop an HPLC method with fluorescence detection for simultaneous quantification of NAC and NACA in biological blood and tissue samples. A gradient HPLC program with fluorescence detection (lambda(ex) = 330 nm, lambda(em) = 376 nm) using N-(1-pyrenyl)maleimide (NPM) as the derivatizing agent was developed. The calibration curves were linear over a concentration range of 25-5000 nm (r(2) > 0.997). The coefficients of variation for within-run precision and between-run precision ranged from 0.67 to 5.23% and for accuracy ranged from 0.98 to 10.54%; the percentage relative recovery ranged from 94.5 to 102.8%. This new method provides satisfactory separation of NAC and NACA, along with other biological thiols, in 20 min with a 5 nm limit of detection (LOD) per 5 microL injection volume.

Effect of hippuric acid on the gaschromatographic retention of S-phenylmercapturic acid.

S-phenylmercapturic acid (PMA) is one specific urinary biomarker of low-level benzene exposure. It is used for biological monitoring of benzene-exposed workers in the petrochemical industry and normally ranges from non-measurable to 10 microg/l levels in non-exposed non-smoking subjects. Benzene-exposure caused by workplace or lifestyle sources is frequently accompanied by toluene exposure, which can cause the occurrence of high levels (from 10 mg/l to more than 2000 mg/l) of hippuric acid (HA) in urine. Both solvents are toxic, and benzene is classified as a human carcinogen. The biological monitoring of benzene and toluene is therefore required for preventive care of exposed workers health. In this study a GC-MS method was adopted for measuring urinary PMA, which involved liquid-liquid extraction (LLE) with ethyl acetate from acidified urine and esterification with 0.5 N hydrochloric acid in methanol. The method evidenced a GC effect in a conventional HP-5 (30 m x 0.25 mm i.d., 0.25 microm film-thickness) methyl-phenylsilicone capillary column produced by HA on PMA. The results demonstrate that HA at concentrations as low as 250 mg/l can delay the elution of PMA and labelled internal standard from the column. The recognition and discussion of this particular GC phase soaking effect may be of help for those who are occupied in the determination of PMA and of urinary acidic metabolites by GC.

A new structural class of proteasome inhibitors identified by microbial screening using yeast-based assay.

A yeast-based growth interference assay was developed utilizing a yeast strain in which expression of Xenopus cyclin A1 was induced to elevate cell division cycle 28 (Cdc28) kinase activity. Since the hyperactivation of Cdc28 kinase in yeast results in a growth-arrest phenotype, compounds which could rescue the cyclin A1-induced growth arrest might be potential new, antitumor drug candidates acting on the cyclin-dependent, kinase-mediated, cell cycle regulation pathway. In the course of our microbial screening program, the new Streptomyces metabolites, belactosins, were identified. As reported previously, belactosin A induced cell cycle arrest at G2/M phase in human cancer cells. However, the molecular mechanism of action was unknown. We herein demonstrate the proteasome inhibition by belactosin A. Belactosin A did not inhibit yeast Cdc28 kinase and human cyclin-dependent kinase in vitro. On the other hand, it inhibited the chymotrypsin-like activity of the rabbit 20S proteasome. From the initial SAR studies, we identified a hydrophobic belactosin A derivative, KF33955, which exhibited a 100-fold greater growth-inhibitory activity against HeLa S3 cells than belactosin A, presumably due to its higher cell permeability. The biochemical analysis using KF33955 suggested that the proteasome inhibitory activity of KF33955 were irreversible and required the beta-lactone moiety to inhibit the proteasome. KF33955 increased the intracellular levels of protein ubiquitination in NIH3T3 cells. In addition, KF33955 treatment resulted in the accumulation of known proteasome substrates in HeLa S3 cells. These results identify belactosin A as a useful lead compound to target proteasome for the treatment of disease whose etiology is dependent on the unregulated ubiquitin-proteasome pathway.

Slowing tumorigenic progression in TRAMP mice and prostatic carcinoma cell lines using natural anti-oxidant from spinach, NAO--a comparative study of three anti-oxidants.

The TRAMP model and human prostatic cancer (PCA) cell lines DU145 and PC3 are useful forchemopreventive studies. We compared the efficacy of 3 anti-oxidants [a water-soluble natural anti-oxidant. NAO (200 mg/kg). found in spinach leaves; epigallocatechin-3 gallate, EGCG (200 mg/kg), a major green tea polyphenol; and N-acetylcysteine, NAC (125 mg/kg)] plus vehicle in slowing spontaneous tumorigenic progression in TRAMP and wild-type male mice. Sacrifices occurred on weeks 5, 9, and 13. Prostatic histopathology and oxidative-stress blood markers were evaluated. Hyperplasias were ranked by a combination of severity grade and distribution (focal, multifocal, and diffuse). The effectivity of each tested compound in reducing the severity/focalness of hyperplasia varied from lobe to lobe. NAO exerted a significant effect on the dorsal and lateral lobes; NAC, on the anterior and ventral lobes, and EGCG, on the ventral lobe. When the most severe hyperplasia in all 4 lobes of TRAMPs was evaluated, only NAO reduced hyperplasia at weeks 9 and 13. Plasma peroxide levels in TRAMPs were reduced following oral administration of NAO or NAC for 13 weeks; EGCG only slightly reduced these levels. In NAO-treated DU 145 and PC3 PCA cells, inhibition of cellular proliferation occurred in a dose-dependent manner, increasing numbers of G1 cells and reducing ROS levels. The anti-oxidative and antiproliferative properties of NAO may explain its efficacy in slowing the spontaneous prostatic carcinogenic process in the TRAMP and its effects in the cell lines.

Synthesis, isolation and identification of glucuronides and mercapturic acids of a novel antiparasitic agent, licochalcone A.

1. Four glucuronic acid conjugates of licochalcone A (Lica), and their metabolites, have been synthesized using rabbit and pig liver microsomes and purified by preparative hplc. 2. The glucuronides were identified as E-Lica 4'-O-beta-glucuronide, E and Z-Lica 4-O-beta-glucuronide and a mono-glucuronide conjugate of a beta-hydroxylated Lica metabolite. The metabolites were identified by hplc-nmr (one and two-dimensional nmr) as well as hplc-ms. 3. At pH 8.5 Lica reacted with N-acetyl-L-cysteine giving the two epimeric conjugates, which were then isolated by preparative hplc and identified by one and two-dimensional nmr spectroscopic methods. 4. Only two glucuronic acid conjugates (E- and Z-Lica 4-O-beta-glucuronide) were found in the urine of rat after i.p. administration of a single dose of Lica.

Biotransformation of allyl chloride in the rat. Influence of inducers on the urinary metabolic profile.

Allyl chloride (AC) is used as intermediate in the synthesis of epichlorohydrin (ECH). We investigated the biotransformation of AC in rats to select potential urinary biomarkers of exposure. For this purpose, we developed analytical methods to measure different selected urinary metabolites of AC. The earlier described urinary metabolites of AC [allyl mercapturic acid (ALMA) and 3-hydroxypropyl mercapturic acid (HPMA)], as well as two urinary metabolites of ECH [alpha-chlorohydrin (alpha-CH) and 3-chloro-2-hydroxypropyl mercapturic acid (CHPMA)], were determined in this study. After intraperitoneal administration of AC, in doses ranging from 66 to 590 mumol/kg, control rats excreted 30 +/- 6.5% of the AC dose as ALMA. HPMA was a minor urinary metabolite of AC (< 3% of the AC dose), and, for this metabolite, no clear dose-excretion relationship was found. Two other minor urinary metabolites were also found as well, namely CHPMA and alpha-CH, suggesting the formation of ECH. CHPMA excretion was linear from 66 to 330 mumol/kg AC and amounted to 0.21 +/- 0.08% of the AC dose. alpha-CH excretion was linear in the dose range used and was excreted for 0.13 +/- 0.02% of the AC dose. In addition, we investigated the influence of three different enzyme inducers on the urinary metabolite profile of AC, namely pyrazole, beta-naphthoflavone, and phenobarbital. Pyrazole only increased the urinary excretion of alpha-CH. beta-Naphthoflavone induction only enhanced the ALMA excretion significantly. Phenobarbital inducted both the excretion of CHPMA and alpha-CH. From these studies, we conclude that urinary excretion of ALMA, CHPMA, and alpha-CH can be used as biomarkers in humans potentially exposed to AC. However, ALMA seems to be the more appropriate biomarker, because enzyme induction may play a confounding role if CHPMA or alpha-CH is used.

Identification and quantification of the N-acetylcysteine conjugate of allyl isothiocyanate in human urine after ingestion of mustard.

Allyl isothiocyanate (AITC) is a constituent of cruciferous vegetables. It occurs widely in the human diet as a natural ingredient or food additive. AITC possesses numerous biochemical and physiological activities. It is cytotoxic and tumorigenic at high doses and also is a modulator of enzymes involved in metabolism of xenobiotics, including carcinogens. It is plausible that the wide consumption of dietary AITC may have profound effects on human health. To facilitate investigations of the effects of dietary AITC in humans, a method of measuring its uptake is needed. In this study, a urinary marker was developed for quantifying AITC uptake in humans. Four adult volunteers were asked to eat a meal containing brown mustard as the source of AITC. The 48-h urine samples were collected from these individuals and analyzed by reverse phase high performance liquid chromatography. A major urinary metabolite was found, which was identified as N-acetyl-S-(N-allylthiocarbamoyl)-L-cysteine, the N-acetylcysteine conjugate of AITC, by comparing its retention time and UV, nuclear magnetic resonance, and mass spectra with those of the synthetic standard. After ingestion of mustard, the AITC conjugate was detected in urine collected from 0 to 12 h. No conjugate was found in urine samples collected after 12 h. The major portion of this metabolite was excreted within 8 h. The average total excretion of AITC conjugate was 5.4 +/- 1.7 (SD) mg after consumption of 10 g of mustard and 12.8 +/- 2.0 mg when 20 g of mustard was consumed. Thus, a dose-dependent excretion of this metabolite was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of enantiomers of N-acetylcysteine by capillary electrophoresis after derivatization by o-phthaldialdehyde.

The optical purity of N-acetyl-L-cysteine was tested by capillary electrophoresis. The D-enantiomer can be found down to a ratio of 0.4:99.6 within 4 min without the use of the D-form or the racemate as a standard. The efficiency was optimized during method development up to a theoretical plate number, N, of several hundred thousand. The migration order and the separation mechanism was explained. The assumed structure of the formed isoindole S-[2-(1-carboxy-2-methylpropyl)isoindole-1-yl]-N-acetylcysteine diastereomers was confirmed and its stability was examined.

Matlystatins, new inhibitors of typeIV collagenases from Actinomadura atramentaria. I. Taxonomy, fermentation, isolation, and physico-chemical properties of matlystatin-group compounds.

During the course of a screening for inhibitors of typeIV collagenases, new metabolites, designated matlystatins, have been isolated from an actinomycete strain, which was identified as a strain of Actinomadura atramentaria. Matlystatins were composed of five congeners, which were separated and purified by n-butanol extraction and chromatography.

Isolation of a mercapturate adduct produced subsequent to glutathione conjugation of bioactivated 3-methylindole.

Bioactivation of the pneumotoxin 3-methylindole (3MI) to a methylene imine intermediate has been demonstrated previously by trapping the electrophile with glutathione in goat lung microsomal incubations. To determine whether the same bioactivation process occurs in whole animals, 3MI was administered to goats, mice, and rats, and the urinary metabolites from these three species were analyzed by HPLC for the presence of the mercapturate that would be expected as the processed and excreted form of the 3MI-glutathione adduct. The mercapturate, 3-[(N-acetylcysteine-S-yl)-methyl]indole (3MI-NAC), was identified in the urine from all three species and was isolated from rat urine for structural identification by uv, NMR, and mass spectrometry. Synthetic 3MI-NAC had uv, NMR, and chromatographic characteristics identical to the isolated metabolite. The presence of this mercapturate in the urine of treated animals unequivocally demonstrates that 3MI is bioactivated to the methylene imine in vivo and that the glutathione adduct is also formed, presumably to detoxify the methylene imine.